The replication of mouse hepatitis virus strain JHM (MHV-JHM), a murine coronavirus, will be studied. Previously isolated and characterized temperature sensitive mutants will be used to investigate the mechanism of virus-specific RNA synthesis. In vitro temperature shift experiments will be performed to identify mutants with defects in negative strand and positive strand RNA synthesis. The defects in RNA synthesis present in these mutants will be further investigated in an in vitro RNA dependent RNA polymerase system. The RNA products of this system will be identified and characterized by gel electrophoresis and hybridization. Antibody made to proteins encoded by defined portions of the 5' region of the MHV genome will also be used to attempt to dissect the mechanism of MHV RNA synthesis. Our RNA positive mutants will also be studied by iso-electric focusing, gel electrophoresis, Western blotting, and electron microscopy in order to identify their temperature sensitive lesions. A persistently infected cell line which has been modulated by antibody will be examined to determine which genes are being expressed. Western and Northern blots will be used to examine MHV-specific RNAs and their expression.